Erythropoietin (hereinafter, also referred to as EPO), an acidic glycoprotein hormone that promotes the differentiation or growth of erythroid progenitor cells, is produced in large amounts as recombinant human EPO by a genetic engineering method. The present applicant has successfully developed a preparation of this purified EPO and introduced, to the market, a product thereof in the form of a freeze-dried or solution preparation as a renal anemia-improving agent or the like.
An EPO solution preparation containing, as a stabilizer, an amino acid selected from leucine, tryptophan, serine, glutamic acid, arginine, histidine, and lysine, and salts thereof, has been proposed as a formulation for introducing a stable EPO solution preparation to the market (Japanese laid-open Patent Publication No. 2000-247903).
The stability of a physiologically active protein solution largely depends on the pH of the solution, and this protein solution is relatively stable at an acidic pH. EPO has been known to be stable at an acidic pH around 6.0. An EPO solution preparation stable even after long-term storage that is currently on the market is a product having a weakly acidic pH of 6.0, which slightly deviates from the in-vivo pH. However, the subcutaneous injection of such a weakly acidic solution preparation is expected to involve pain. The development of an EPO solution preparation that is further improved in usability and is stable even after long-term storage has been demanded.
The addition of saccharides such as mannitol has been proposed as a method for alleviating pain involved in the administration of a preparation for injection (WO 02/11753).
Alternatively, an antibody preparation containing a poloxamer as a surfactant has been reported (WO 2004/075913). However, this document has made no description of the stability of a solution preparation containing EPO or pain involved in injection.
An object of the present invention is to provide an EPO solution preparation which alleviates pain involved in injection and is stable even after long-term storage. A further object of the present invention is to provide a method for quantifying a protein in a trace amount, which is useful both for the quantification of the erythropoietin-containing solution preparation and for a purity test.